Hypertension affects 60 million adults in the United States, and the insulin resistance syndrome induces a salt- sensitive form of hypertension in approximately half of these individuals. In the insulin resistance syndrome compensatory hyperinsulinemia provoked by peripheral resistance increases sodium retention and hence, blood pressure via direct effects on the renal tubule. The epithelial sodium channel (ENaC) expressed in principal cells of the collecting duct is fundamental to this process. However, the receptors in principal cells which propagate insulin-induced ENaC stimulation in the insulin resistance syndrome are not established. Experimental studies have yielded conflicting results on the role of the insulin receptor in mediating renal sodium reabsorption. On the other hand, several studies have suggested that activation of the insulin-like growth factor-1 (IGF-1) receptor can enhance sodium reabsorption via ENaC. The objective in this proposal is to determine the mechanisms that specifically regulate ENaC activity in models of insulin resistance. The R01 Grant will provide the necessary resources for the principal investigator (PI) to test the hypothesis that hyperinsulinemia provoked by peripheral insulin resistance induces salt-sensitive hypertension via activation of the insulin and/or the IGF-1 receptor, initiation of downstream signaling cascades, and enhancement of ENaC- mediated sodium transport. To test this hypothesis, two specific aims are proposed. Aim 1 is to define the contribution of the insulin and/or IGF-1 receptor to blood pressure, sodium reabsorption, and ENaC-mediated sodium transport in mouse models of insulin resistance. Using wild-type and principal cell-specific insulin or IGF-1 receptor knockout mice, the PI will induce insulin resistance in these mice and then measure systemic blood pressure, urine sodium excretion in the presence and absence of an ENaC-specific inhibitor, and sodium transport across in vitro microperfused collecting duct. Aim 2 is to determine the cellular mechanisms by which insulin-mediated activation of the insulin and/or IGF-1 receptor stimulates ENaC-mediated sodium transport in principal cells. The PI will measure receptor activation in control vs. insulin-resistant wild-type mice;measure ENaC expression in insulin-resistant wild-type vs. knockout mice;and compare ENaC-mediated currents and receptor signaling pathways using a novel technique to isolate principal cells from wild-type and knockout mice. The expected outcomes of the proposed aims are to identify the receptors and post-receptor signaling pathways required for stimulation of ENaC and hence, salt-sensitive hypertension in the insulin resistance syndrome. We anticipate that these results will significantly advance the field by identifying appropriate targets for the prevention and treatment of hypertension in diseases associated with insulin resistance, such as obesity and Type 2 diabetes mellitus. The proposed research is innovative in that we will directly compare the insulin and IGF-1 receptor and employ novel techniques to challenge the unilateral paradigm of hyperinsulinemia simply activating the insulin receptor in ENaC-expressing principal cells of the collecting duct. PUBLIC HEALTH RELEVANCE: As the incidence of insulin resistance increases in the general population, so will the incidence of hypertension and cardiovascular disease. The kidney is insulin-sensitive while other organs are insulin-resistant;thus, in the insulin resistance syndrome high plasma levels of insulin stimulate sodium reabsorption in kidney collecting duct and induce a salt-sensitive form of hypertension. The mechanisms of how insulin stimulates sodium reabsorption are not established, and by studying these mechanisms in insulin-resistant mice, we can learn more about how insulin resistance causes salt-sensitive hypertension and how to design specific therapies for this subset of individuals.